Human cytomegalovirus (HCMV) is the herpesvirus of greatest public health and medical significance in the US. In addition to its societal importance as a human pathogen, the aspects of human biology learned by the virus during its co-evolution with humans have enabled development of HCMV into an extraordinarily valuable model microbe that has helped to reveal many aspects of virus-host interactions in the areas of immunology, molecular biology, and cell biology. The HCMV cytoplasmic virion assembly complex (cVAC) is a structure about the size of a nucleus where developing virions acquire most of their tegument, are enveloped, and are then transported to the cell surface for release. cVAC biogenesis involves dramatic cytoplasmic remodeling that takes place during the first 2 to 4 days after infection. We found that the cVAC is arranged as a set of nested cylinders, with the outer cylinder consisting of networks of tubular vesicles derived from the Golgi apparatus and the trans-Golgi network; the inner cylinder consists of vesicles derived from recycling endosomes where virions are enveloped and then transported to the cell surface. From quantitative analyses of the distribution of markers for various components of the secretory and endosomal machinery, we identified striking examples of HCMV-induced shifts in the identities of organelles involved in protein transport, and we have identified three HCMV genes as candidate regulators of cVAC biogenesis. Our central hypothesis is that HCMV proteins orchestrate creation of the cVAC via interactions of viral proteins with cellular proteins and organelles to optimize infectious virion production and egress. To test this hypothesis, in Aim 1, we will define the roles of the virus genes we have identified as candidate regulators of cVAC biogenesis and use them as probes to delineate the molecular pathway that results in assembly of the cVAC. In Aim 2, we will determine how the reorganized secretory organelles and machinery contribute to the process of virion assembly and maturation. We will employ recombinant viruses to express candidate viral and cellular regulatory proteins that have been engineered to contain protein destabilization domains, enabling either destruction or restabilization of the protein of interest at desired times during cVAC biogenesis and virus replication. We will then use a battery of assays to measure the effects on virus replication, and to compare the functions of the affected organelles and machinery with and without inactivation of the regulator of interest. Our studies will result in (i) illumination of the process of virion maturation and how HCMV manages cellular systems for its benefit, (ii) definition of new targets for development of novel antiviral compounds, and (iii) new insights into the mechanisms that regulate the biogenesis of cellular protein transport and processing organelles.